Papers by Ramakrishna V Hosur
Journal of Physical Chemistry B, May 28, 2019
Intrinsically disordered proteins (IDPs) form a special category because they lack a unique well-... more Intrinsically disordered proteins (IDPs) form a special category because they lack a unique well-folded 3D structure under physiological conditions. They play crucial role in cell signaling, regulatory functions and responsible for several diseases. Although, they are abundant in nature, only a small fraction of it has been characterized till date. Such proteins adopt a range of conformations and can undergo transformation from disordered-to-ordered state or vice-versa upon binding to ligand. Insights of such conformational transition is perplexing in several cases. In the present study, we characterized disordered as well as ordered states and the factors contributing the transitions through a mutational study by employing replica exchange molecular dynamics simulation on a βγ-crystallin. Most of the proteins within this superfamily are inherently ordered. However, Hahellin, although a member of βγcrystallin, it is intrinsically disordered in its apo-form which takes a well-ordered βγ-crystallin fold upon binding to Ca 2+. It is intriguing that the mutation at the 5 th position of the canonical motif to Arg increases the domain stability in several ordered microbial βγ-crystallins with concomitant loss in Ca 2+ binding affinity. We carried out similar Ser to Arg mutations at 5 th position of the canonical motif for the first time in an intrinsically disordered protein to understand the mechanistic insights of conformational transition. Our study revealed that newly formed ionic and hydrogen bonding interactions at the canonical Ca 2+ binding sites play crucial role in transforming the disordered conformation into ordered βγ-crystallin. .
RSC Advances, 2022
The accumulation of the intrinsically disordered protein alpha-synuclein (aSyn) in the form of in... more The accumulation of the intrinsically disordered protein alpha-synuclein (aSyn) in the form of insoluble fibrillar aggregates in the central nervous system is linked to a variety of neurodegenerative disorders such as Parkinson's disease, Lewy body dementia, and multiple system atrophy. Here we show that Pyrogallol, Corilagin and Chebulagic acid, compounds containing a different number of catechol rings, are independently capable of delaying and reducing the extent of aSyn fibrillization. The efficiency of inhibition was found to correlate with the number of catechol rings. Further, our NMR studies reveal that these compounds interact with the N-terminal region of aSyn which is unstructured even in the fibrillar form of the protein and is known as the "fuzzy coat" of fibrils. Thus, Corilagin and Chebulagic acid target the fuzzy coat of aSyn and not the amyloid core which is a common target for the inhibition of protein fibrillization. Our results indicate that the N-terminus also plays a key role in the fibrillization of aSyn.
NMR structural studies on a bacterially produced DNA binding domain of Drosophila c-Myb protein a... more NMR structural studies on a bacterially produced DNA binding domain of Drosophila c-Myb protein as well as its cognate DNA sequence carried out in our laboratory have been surveyed. The structure of a self-complementary dodecamer DNA containing the Myb responsive element (TAACGG) has been determined to atomic resolution by the combined use of two-dimensional NMR, spectral simulations, restrained energy minimization and distance geometry calculations. The structure is seen to possess novel features which may play important roles during its interaction with the Myb protein, The DNA binding domain of c-Myb protein was seen to have a hydrophobic core and we have identified the types of residues contributing to its formation. Residues contributing to the hydrophobic core formation are seen to be well spread out over the whole length of the 160 residues in the protein and include isoleucines, valines, leuclnes, alanines threonines, aromatic residues, glutamines and possibly aspartates. Ou...
Journal of Magnetic Resonance (1969), 1986
ABSTRACT
Journal of Magnetic Resonance (1969), 1985
Journal of Biomolecular Nmr, 2001
Two triple resonance experiments, HNN and HN(C)N, are presented which correlate HN and 15N resona... more Two triple resonance experiments, HNN and HN(C)N, are presented which correlate HN and 15N resonances sequentially along the polypeptide chain of a doubly (13C, 15N) labeled protein. These incorporate several improvements over the previously published sequences for a similar purpose and have several novel features. The spectral characteristics enable direct identification of certain triplets of residues, which provide many starting
Biomolecular NMR Assignments, 2010
The GTPase effector domain (GED) is a subunit of dynamin, a multi-domain protein involved in endo... more The GTPase effector domain (GED) is a subunit of dynamin, a multi-domain protein involved in endocytosis. GED forms a megadalton-sized self-assembly in vitro. The core of such huge assemblies is inaccessible to detailed Nuclear Magnetic Resonance characterization by conventional methods due to line broadening effects. Till date, there have been no studies to directly identify the residues involved in the core of the assembly. In this background we report here the NMR resonance assignments of deuterated dimethyl sulfoxide (DMSO-d6)denatured GED from Homo sapiens. This will form the basis for probing the core of GED assembly and characterization of the association pathway driven by DMSO dilution.
Journal of Magnetic Resonance (1969), 1988
The phase characteristics of two chemical-shift scaling pulse sequences COSS and S.COSY have been... more The phase characteristics of two chemical-shift scaling pulse sequences COSS and S.COSY have been analyzed, and a method for eliminating dispersive contributions to cross and diagonal peaks in the 2D spectra has been suggested. This results in pure absorption peak shapes for both diagonal-and cross-peak components and both acquire in-phase characteristics. An experimental demonstration of the procedure is given with a onedimensional simplification due to practical limitations of the available 2D software. 8 1988 Aadnnic PITS, Inc.
Biomolecular Forms and Functions, 2013
European Journal of Biochemistry, 2004
The NMR structure of 2′,5′ d(GGGGCCCC) was determined to gain insights into the structural differ... more The NMR structure of 2′,5′ d(GGGGCCCC) was determined to gain insights into the structural differences between 2′,5′‐ and 3′,5′‐linked DNA duplexes that may be relevant in elucidating nature's choice of sugar‐phosphate links to encode genetic information. The oligomer assumes a duplex with extended nucleotide repeats formed out of mostly N‐type sugar puckers. With the exception of the 5′‐terminal guanine that assumes the syn glycosyl conformation, all other bases prefer the anti glycosyl conformation. Base pairs in the duplex exhibit slide (−1.96 Å) and intermediate values for X‐displacement (−3.23 Å), as in ADNA, while their inclination to the helical axis is not prominent. Major and minor grooves display features intermediate to A and BDNA. The duplex structure of iso d(GGGGCCCC) may therefore be best characterized as a hybrid of A and BDNA. Importantly, the results confirm that even 3′ deoxy 2′,5′ DNA supports duplex formation only in the presence of distinct slide (≥−1.6 Å) ...
Journal of Magnetic Resonance, 2010
Time-saving in data acquisition is a major thrust of NMR pulse sequence development in the contex... more Time-saving in data acquisition is a major thrust of NMR pulse sequence development in the context of structural proteomics research. The conventional HNCA and HN(CA)CO pulse sequences, routinely used for sequential backbone assignment, have the limitation that they cannot distinguish inter- and intra-residue correlations. In order to remove this ambiguity, one has to record HNCO and HN(CO)CA or sequential HNCA experiments which provide unambiguous information of sequential correlations. However, this almost doubles the experimental time. Besides, they require repeated scanning through the (15)N planes to search for the matching peaks along the carbon dimension. In this background, we present here two pulse sequences, termed as hncoCANH and hnCOcaNH that lead to spectra equivalent to HNCA and HN(CA)CO spectra, respectively, but with direct distinction of inter- and intra-residue peaks; these occur with opposite signs in the new experiments. The two pulse sequences have been derived by simple modification of the previously described HN(C)N pulse sequence [Panchal et al., J. Biomol. NMR 20 (2001) 135-147] to frequency-label (13)C(alpha) or…
Journal of Biomolecular NMR, 1991
An efficient algorithm for generating DNA structures from a given set of distance constraints has... more An efficient algorithm for generating DNA structures from a given set of distance constraints has been developed. The present implementation is suited for single-stranded DNA. The performance of the program has been tested with constraint sets representative of most stringent theoretical cases and also with usually available experimental ones. The results indicate that use of NOE-derived constraints alone generates an extremely large family of conformers and suggest that the quality of structure determination may be enhanced by incorporating additional constraints obtained by other means. The speed of the program makes it ideal for interactive use in conjunction with other complementary algorithms such as those for spectral simulation, energy minimization and molecular dynamics calculations.
Journal of Biomolecular NMR, 2009
Understanding protein stability at residue level detail in the native state ensemble of a protein... more Understanding protein stability at residue level detail in the native state ensemble of a protein is crucial to understanding its biological function. At the same time, deriving thermodynamic parameters using conventional spectroscopic and calorimetric techniques remains a major challenge for some proteins due to protein aggregation and irreversibility of denaturation at higher temperature values. In this regard, we describe here the NMR investigations on the conformational stabilities and related thermodynamic parameters such as local unfolding enthalpies, heat capacities and transition midpoints in DLC8 dimer, by using temperature dependent native state hydrogen exchange; this protein aggregates at high (>65 degrees C) temperatures. The stability (free energy) of the native state was found to vary substantially with temperature at every residue. Significant differences were found in the thermodynamic parameters at individual residue sites indicating that the local environments in the protein structure would respond differently to external perturbations; this reflects on plasticity differences in different regions of the protein. Further, comparison of this data with similar data obtained from GdnHCl dependent native state hydrogen exchange indicated many similarities at residue level, suggesting that local unfolding transitions may be similar in both the cases. This has implications for the folding/unfolding mechanisms of the protein.
Biophysical Chemistry, 1997
The oncogene product c-myb is a transcriptional modulator and is known to play important roles in... more The oncogene product c-myb is a transcriptional modulator and is known to play important roles in cell growth and differentiation. It binds to DNA in a sequence specific manner and its cognate sequence motifs have been detected in the genes of proteins implying its role in a variety of regulatory functions. The protein has a DNA binding domain consisting of three imperfect repeats with highly conserved tryptophans at regular spacings in each of the repeats. We have carried out a variety of investigations on the structure and interactions of the DNA binding domain of Drosophila c-myb and its cognate DNA target sequences. The domain has been bacterially over-expressed by subcloning a segment of the gene coding for the domain in a pET 11d vector and transforming it into E. coli BL21 (DE3). Circular dichroism of the protein has revealed that the domain is largely helical in nature. Fluorescence investigations indicated that three out of the nine tryptophans are solvent exposed and the others are buried in the interior. The recombinant protein is able to distinguish between specific and non-specific DNA targets in its binding and the interaction is largely electrostatic in nature in both cases. Dynamic fluorescence quenching experiments suggested that the DNA binding sites on the protein for specific and non-specific DNA targets are physically different. Most of the conserved tryptophans are associated with the specific DNA binding site. Simulated annealing and molecular dynamic simulations in a water matrix have been used to predict an energetically favoured conformation for the protein. Calculation of surface accessibilities of the individual residues shows that nearly 60% of the residues are less than 50% accessible to the solvent. Two and three dimensional NMR experiments with isotopically labelled protein have enabled spin system identification for many residue type and the types of residues involved in hydrophobic core formation in the protein. In an attempt to see the DNA surface possibly involved in specific interaction with the protein, a three-dimensional structure of a 12 mer cognate DNA has been determined by NMR in conjunction with restrained energy minimization. The recognition sequence shows interesting structural characteristics that may have important roles in specific interaction.
Biophysical Chemistry, 2008
Characterization of the low energy excited states on the energy landscape of a protein is one of ... more Characterization of the low energy excited states on the energy landscape of a protein is one of the exciting and challenging problems in structural biology today. In this context, we present here residue level NMR description of the low energy excited states representing locally different alternative conformations in the dynein light chain protein, in its dimeric as well as monomeric forms. Important differences have been observed between the two cases and these are not necessarily restricted to the dimer interface. Simulations indicate that the low energy excited states are within a free energy of 2-3 kcal/mol above the native state. In both the monomer and the dimer the energy landscape is very sensitive to small pH perturbations. Nearly 25% of the residues (total of residues at pH 3.0 and 3.5 for the monomer, and at pH 7.0 and 6.0 for the dimer) access alternative conformations. The observations have been rationalized on the basis of protonation-deprotonation equilibria in the side chains; histidines in the case of the dimer and aspartates/glutamates in the case of the monomer. The possible relationship of the observed ruggedness of the native energy landscape with the protein structure, and its implications to protein adaptability and unfolding have been discussed.
Biochemistry, 1994
The N-terminal DNA-binding domain of c-myb oncoprotein binds to D N A in a sequencespecific manne... more The N-terminal DNA-binding domain of c-myb oncoprotein binds to D N A in a sequencespecific manner. The domain, consisting of three imperfect tandem repeats, has tryptophan residues at very regular intervals and this is believed to be of some significance in the DNA-binding activity of the protein. We have found that the hydrophobic-site-specific probe 6-p-toluidino-2-naphthalenesulfonate (TNS) binds to the bacterially expressed DNA-binding domain of Drosophila c-myb protein (R123). TNS has a single binding site on this protein with an apparent dissociation constant in the range of (5-8) X lo-' M. When the TNS-protein complex was treated with an oligomeric D N A duplex having a cognate myb-binding site, the T N S was displaced from the complex. Nonspecific D N A duplex oligomers were ineffective, indicating that T N S displacement was a sequence-specific process. W e examined further some features of the TNSbinding site on the protein, taking advantage of the fluorescence properties of the protein and the bound TNS. Our data indicate that the T N S binding occurs in a peripheral site on the protein in a manner that allows the bound T N S to be solvent accessible. Furthermore, there are indications that tyrosine(s) and tryptophans of the protein mediate resonance energy transfer to the bound TNS. From fluorescencequenching data of the protein and protein-TNS complex, we could assess that both solvent-accessible and internal tryptophans are in the vicinity of the bound TNS. Our data are consistent with the proposal that
Biochemistry, 1993
Solution conformation of a self-complementary 1Cmer D N A duplex (d-GGATTGGCCAATCC) containing th... more Solution conformation of a self-complementary 1Cmer D N A duplex (d-GGATTGGCCAATCC) containing the GCCAAT recognition motif of several transcription factors has been investigated by N M R spectroscopy. Complete resonance assignment of all the protons (except H5',H5" protons) has been obtained following standard procedures based on two-dimensional N M R techniques. Three-bond coupling constants have been determined by spectral simulation procedures. New strategies have been described and employed for quantifying N O E intensities from the structural point of view. Approximate ranges of y torsion angles have been obtained from a selective NOESY experiment, by estimating the J(4'-5'), J(4'-5''), or their sum in the Hl'-H4' cross peaks of the spectrum. Likewise, ranges of c torsion angles have been obtained by monitoring the H3' multiplicities in the H8/H6-H3' cross peaks in selective NOESY spectra. With the help of such a total of 73 coupling constraints, 79 N O E intensity constraints, and 108 H-bond constraints, model building has been carried out to obtain a structure which satisfies the constraints. Starting from I Abbreviations: COSY, correlated spectroscopy; NOE, nuclear Overhauser effect; NOESY, NOE correlated spectroscopy; TANDY, torsion angle approach to nucleic acid distance geometry; CORMA, complete relaxation matrix analysis; COMATOSE, complete matrix analysis torsion optimized structure; MARDIGRAS, matrix analysis of relaxation for discerning geometry of an aqueous structure; IRMA, iterative relaxation matrix approach; SICOS, simulation of correlated spectra; BURP, band-selective uniform response and phase; DG, distance geometry; RMSD, root mean square deviation.
Biochemistry, 2001
Sequence specific resonance assignment is the primary requirement for all investigations of prote... more Sequence specific resonance assignment is the primary requirement for all investigations of proteins by NMR methods. In the present postgenomic era where structural genomics and protein folding have occupied the center stage of NMR research, there is a high demand on the speed of resonance assignment, whereas the presently available methods based either on NOESY or on some triple-resonance experiments are rather slow. They also have limited success with unfolded proteins because of the lack of NOEs, and poor dispersion of amide and carbon chemical shifts. This paper describes an efficient approach to rapid resonance assignment that is suitable for both folded and unfolded proteins, making use of the triple-resonance experiments described recently [HNN and HN(C)N]. It has three underlying principles. First, the experiments exploit the 15 N chemical shift dispersions which are generally very good for both folded and unfolded proteins, along two of the three dimensions; second, they directly display sequential amide and 15 N correlations along the polypeptide chain, and third, the sign patterns of the diagonal and the sequential peaks originating from any residue are dependent on the nature of the adjacent residues, especially the glycines and the prolines. These lead to so-called "triplet fixed points" which serve as starting points and/or check points during the course of sequential walks, and explicit side chains assignment becomes less crucial for unambiguous backbone assignment. These features significantly enhance the speed of data analysis, reduce the amount of experimentation required, and thus result in a substantially faster and unambiguous assignment. Following the amide and 15 N assignments, the other proton and carbon assignments can be obtained in a straightforward manner, from the well-established three-dimensional triple-resonance experiments. We have successfully tested the new approach with different proteins in the molecular mass range of 10-22 kDa, and for illustration, we present here the backbone results on the HIV-1 protease-tethered dimer (molecular mass ∼ 22 kDa), both in the folded and in the unfolded forms, the two ends of the folding funnel. We believe that the new assignment approach will be of great value for both structural genomics and protein folding research by NMR.
Biochemical and Biophysical Research Communications, 2000
We report the NMR structure of the DNA sequence d-TGGGCGGT in Na ؉ solutions at neutral pH, conta... more We report the NMR structure of the DNA sequence d-TGGGCGGT in Na ؉ solutions at neutral pH, containing a repeat sequence from SV40 viral genome. The structure is a novel quadruplex incorporating the C-tetrad formed by symmetrical pairing of four Cs via NH 2 OO 2 H-bonds in a plane. The C-tetrad has a wider cavity compared to G-tetrads and stacks well over the adjacent G4-tetrad, but poorly on the G6 tetrad. The quadruplex helix is largely underwound by 8-10°compared to B-DNA except at the C5-G6 step. To our knowledge this is the first report of C-tetrad formation in DNA structures, and would be of significance from the point of view of both structural diversity and specific recognition.
Biochemical and Biophysical Research Communications, 2000
Higher ordered structures formed by different DNA sequences have been widely investigated in rece... more Higher ordered structures formed by different DNA sequences have been widely investigated in recent years because of their implications in a variety of biological functions. Among these, G-quadruplexes have exhibited a great variety depending on the exact sequence, the lengths of the G-stretches, interception by other nucleotides, and environmental conditions such as pH, temperature, salt type, and its concentration. We report here interesting conformational switches observed by NMR in the sequence d-TGGCGGC containing two GGC triplet repeats related to the disease Fragile X-Syndrome. At neutral pH, the solution structure is a parallel-stranded quadruplex in presence of K ؉ ions. Lowering the pH does not cause a major change in the structure; however, the chemical shift patterns of the C4 and G3 base protons suggest protonation of the C-tetrad in the center of the quadruplex. In contrast, the sequence forms an antiparallel duplex in Na ؉ containing solutions. As the pH of the Na ؉ sample is lowered, an equilibrium mixture of a duplex and a quadruplex appears, and at pH 2.2, the molecule exists entirely as a quadruplex. These results would be of significance from the point of view of recognition and regulation by different helicase enzymes, which have been found to discriminate between different types of quadruplex structures.
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Papers by Ramakrishna V Hosur